U.S. patent number 5,487,895 [Application Number 08/106,342] was granted by the patent office on 1996-01-30 for method for forming controlled release polymeric substrate.
This patent grant is currently assigned to Vitaphore Corporation. Invention is credited to Gregory S. Dapper, Ronald K. Yamamoto.
United States Patent |
5,487,895 |
Dapper , et al. |
January 30, 1996 |
**Please see images for:
( Certificate of Correction ) ** |
Method for forming controlled release polymeric substrate
Abstract
A method for forming a controlled release polymeric substrate is
provided by contacting a polymeric substrate with a liquid mixture
containing a cross-linking agent at least partially soluble therein
comprising water and an organic liquid, for a period of time and at
a temperature and concentration of the agent sufficient for the
agent to penetrate the substrate to form cross-linking bridges in
the substrate in a decreasing concentration gradient beneath the
surface.
Inventors: |
Dapper; Gregory S. (Newark,
CA), Yamamoto; Ronald K. (San Francisco, CA) |
Assignee: |
Vitaphore Corporation
(Plainsboro, NJ)
|
Family
ID: |
22310898 |
Appl.
No.: |
08/106,342 |
Filed: |
August 13, 1993 |
Current U.S.
Class: |
424/278.1;
424/184.1; 424/484; 424/499; 514/773; 514/774; 514/776; 530/354;
530/356; 530/409 |
Current CPC
Class: |
A61K
9/1658 (20130101); A61K 9/5052 (20130101) |
Current International
Class: |
A61K
9/16 (20060101); A61K 9/50 (20060101); A61K
039/00 (); A61K 009/52 () |
Field of
Search: |
;530/356,354
;424/484,499,184.1,278.1 ;514/774,776,773 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0210461 |
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Jul 1986 |
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EP |
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0367590 |
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Nov 1989 |
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EP |
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91918644 |
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Nov 1993 |
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EP |
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8904668 |
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Jun 1989 |
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WO |
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9110446 |
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Jul 1991 |
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WO |
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Other References
Horakova, Z., et al. "Prolongation of Various Pharmacological
Effects by Means of Collagen Substances", Therapie, vol. 22, 1967,
pp. 1455-1460 and Translation from French. .
M. Chvapil, R. Kronenthal, W. van Winkle; Medical and Surgical
Applications of Collagen; Int. Rev. Conn. Tissres, 6:1-61 (1973).
.
M. Dunn, T. Miyata, K. Stenzel, A. Rubin; Studies on Collagen
Implants in the Vitreous; Surgical Forum/Ophthalmic Surgery, vol.
19, 492-494 (1968). .
A. Rubin, K. Stenzel; Collagen as a Biomaterial; Technology Review,
vol. 71, No. 2, 44-49 (1968). .
E. Balazs, D. B. Sweeney; A. McPherson (ed.); The Injection of
Hyaluronic Acid and Reconstituted Vitreous into the Vitreous
Cavity; New and Controversial Aspects of Retinal Detachment,
36:371-376 (1968). .
S. Richard-Blum, G. Ville; Minireview: Collagen Crosslinking; Int.
J. Biochem., vol. 21, No. 11, 1185-1189 (1989). .
M. Chvapil, D. Speer, W. Mora, C. Eskelson; Effect of Tanning Agent
on Tissue Reaction to Tissue Implanted Collagen Sponge; Journal of
Surgical Research, 35, 402-409 (1983). .
K. Weadock, R. Olson, F. Silver; Evaluation of Collagen
Crosslinking Techniques; Biomat., Med., Dev., Art. Org., 11 (4),
293-318 (1983-84)..
|
Primary Examiner: Kim; Kay K. A.
Attorney, Agent or Firm: Fish & Richardson
Claims
What is claimed is:
1. A method of forming a controlled release polymer substrate
comprising the steps of:
a) providing a water-swellable polymeric substrate;
b) contacting said substrate, or a portion thereof, with a liquid
mixture containing a cross-linking agent which is at least
partially soluble in said liquid mixture comprising water and an
organic liquid, for a period of time and at a temperature and
concentration of said agent sufficient for said agent to penetrate
a surface of said substrate in contact with said mixture, to form
cross-linking bridges in said substrate in a decreasing
concentration gradient beneath said surface;
c) contacting said substrate with a bioactive agent which is
absorbed into said substrate.
2. A method according to claim 1 wherein said substrate comprises a
film and one surface of said film is contacted with said mixture in
step (b).
3. A method according to claim 1 wherein said substrate comprises
droplets of a dispersion of a polymeric material, whereby upon said
contacting in said step (c), said substrate is formed into hollow
spheres.
4. A method according to claim 1 wherein said period of time,
temperature and concentration are selected to form a crosslinked
skin on said substrate.
5. A method for releasing a bioactive agent through a first surface
of a polymer substrate in a controlled manner into body tissue or
fluid comprising the step of contacting said tissue or fluid with
said surface of said substrate loaded with said bioactive agent;
said substrate characterized by a decreasing concentration gradient
of cross-linking bridges beneath a second surface thereof wherein
said bioactive agent is selectively released from said substrate
primarily through said first surface of said substrate wherein said
second surface is proximal to the end of said gradient having a low
concentration of said cross-linking bridges.
6. A method for time-releasing a bioactive agent into body fluid or
tissue comprising the step of contacting said fluid or tissue with
particles comprising a water-swellable polymeric material, said
microspheres impregnated with said bioactive material, wherein said
particles are characterized by a decreasing concentration gradient
of cross-linking bridges from the outer surface toward the interior
such that upon contact of said particles with said body fluid or
tissue, said particles release said bioactive agent.
7. A method according to claim 6 wherein such particles further are
impregnated with an active agent which acts upon said polymeric
material to erode said particles from the interior thereof, whereby
upon contact of said particles with said body fluid or tissue said
active agent commences erosion of said particles whereby the rate
of said erosion is higher at areas of low density of cross-linking
bridges in the interior of said particles and the last portion of
said particles to erode is at the surface of said particles where
the density of cross-linking bridges is greatest.
8. A method according to claim 6 or 7 wherein said particles
comprise hollow spheres.
9. A method according to claim 1 wherein said substrate is selected
from the group consisting of collagen, gelatins, elastins, albumins
and chitosans.
10. A method according to claim 9 wherein said substrate is
selected from the group consisting of natural and modified
collagens.
11. A method according to claim 1 wherein said cross-linking agent
is selected from the group consisting of
1-ethyl-3-(3-diethylaminopropyl carbodiimide and
1-cyclohexyl-3-(2-(3'-morpholino)ethyl) carbodiimide.
12. A method according to claim 1 wherein said organic liquid is
selected from the group consisting of ketones, alcohols, esters and
mixtures thereof.
13. A method according to claim 12 wherein said organic liquid is
selected from the group consisting of methanol, ethanol, acetone,
methylethyl ketone, ethyl acetate, and mixtures thereof.
14. A method according to claim 1 wherein said organic liquid
comprises a water-immiscible solvent.
15. A method according to claim 1 wherein said liquid mixture
comprises from 5% to 95% organic solvent and 95% to 5% water by
volume.
16. A method according to claim 11 wherein said concentration of
said crosslinking agent initially in said liquid mixture is in the
range of 0.1 to 10 mg/ml.
17. A method according to claim 1 wherein period of time of contact
of said substrate with said liquid mixture is in the range of 0.05
to 24 hours.
18. A method according to claim 1 wherein said temperature is in
the range of 4.degree. C. to 50.degree. C.
19. A method according to claim 1 wherein said bioactive agent is
selected from the group consisting of antibiotics, analgesics,
anti-inflammatory agents, anti-glaucoma agents, vaccines, and
anti-neoplastic agents.
20. A method according to claim 1 wherein said step (c) precedes
said step (b).
21. A cross-linked controlled-release polymer substrate containing
a bioactive agent prepared according to any one of claims 1 through
4 or 9 through 20.
22. A method according claim 5 wherein said substrate comprises
collagen crosslinked with 1-ethyl-3-(3-diethylaminopropyl)
carbodiimide.
23. A method according to claim 6 or 7 wherein said polymeric
material comprises collagen crosslinked with
1-ethyl-3-(3-diethylaminopropyl) carbodiimide.
Description
FIELD OF THE INVENTION
The present invention relates to a method for forming controlled
release polymeric substrates which release bioactive materials in a
time-release fashion, by diffusion through the substrate and/or
bioerosion of the substrate. The invention is particularly directed
to the use of collagen or collagen-like substrates which are
cross-linkable.
BACKGROUND OF THE INVENTION
It is desirable in a great number of biomedical fields to be able
to provide controlled release of bioactive agents into body tissues
or fluids. However, due to the multiplicity of the types of
chemical structures of bioactive agents, the desired time-release
profile for a particular bioactive agent, and the diffusion rates
in available and desirable substrates and/or bioerosion of such
substrates in bodily fluids, it can be difficult to determine the
proper combination of substrate/bioactive agent for a suitable
time-release profile, if any. In addition, it would be desirable in
some cases to have a substrate in a particular shape, such as a
lens for use as an ocular insert for treatment of corneal
transplant trauma.
It is thus an object of the present inventions to provide a method
for making collagen and collagen-like substrates which are adapted
through cross-linking for time-release profiles of bioactive
agents.
It is a particular object of the present invention to provide
ocular inserts whereby the outer surface of the ocular insert is
protected against excessive dehydration by cross-linking while the
inner surface of the insert is relatively uncrosslinked to allow
for the release of the bioactive agent into the tear fluid.
These and other objects of the invention will be apparent from the
following description of the invention, the appended claims and
from practice of the invention.
BACKGROUND OF THE INVENTION
U.S. Pat. No. 4,535,010 to Axen, issued Aug. 13, 1985, discloses a
method for coating a solid body or substrate with polymeric layers
which are formed by cross-linking an organic substance. The primary
object of that invention is to coat a substrate, such as plastics,
for example, Plexiglas.RTM. (polymethylmethacrylate), polystyrene,
polyvinyl alcohol, polyvinyl chloride and polyethylene. Porous
substrates are also disclosed such as gel chromatographic media,
for example, Sephadex.RTM., crosslinked dextran and Sepheron.RTM.,
crosslinked poly(hydroxyalkylmethacrylate). The substrate is first
pre-impregnated with a component which is necessary for the
cross-linking reaction which, when contacted with a solution, tends
to diffuse from the substrate into the solution. If the solution
contains a component which prevents the cross-linking reaction from
occurring when the component diffuses from the substrate, the
cross-linking can only take place in the boundary layer of the
solution and the substrate, thereby forming a coating over the
substrate. Alternatively, the substrate may be impregnated with a
modulator (such as hydroxyl ions) which diffuses into the solution.
At the boundary layer between solution and substrate the modulator
contacts the other cross-linking components in the solution thus
causing the cross-linking condition to take place at the boundary
layer. The disclosure in this patent does not appear to provide a
cross-linking gradient into the substrate beneath the surface nor
does it appear to disclose a method for entrapping a bioactive
agent within the substrate to control release therefrom.
U.S. Pat. No. 4,981,912 to Kurihara, issued Jan. 1, 1991, discloses
a method for forming shaped articles of a crosslinked elastomer
having a crosslinked density continuously decreasing from the
surface toward the interior at a specific gradient. The articles
are characterized by surface non-stickiness and low friction
properties while maintaining tensile strength, elongation and
compression set resistant properties as well as elastomeric
properties. The purpose of this invention is apparently to modify
the surface characteristics of the elastomeric substrate while
maintaining or improving other bulk properties.
The elastomeric substrates which are disclosed include natural
rubber, polybutadiene, styrene-butadiene copolymer, polychloroprene
rubber, ethylene propylene copolymer, ethylene-propylene-diene
terpolymer and various other isomeric substrates such as silicone
rubber, urethane rubber, acrylic rubber, fluorosilicone rubber and
the like, all of which are non-water soluble. The elastomer is
first vulcanized using a cross-linking agent which partially
crosslinks the elastomer at a substantially uniform cross-linking
density throughout the entire body of the substrate. Sulfur and
sulfite compounds, oximes, and quinones are disclosed as being
useful for this purpose. The surface of the vulcanized elastomer is
then treated with a second cross-linking agent to provide a second
partial crosslinked density at the surface of the substrate. The
second partial crosslinked density, however, decreases continuously
from the surface toward the interior of the substrate. The second
cross-linking agents are disclosed to be such compounds as the
sulfite compounds, phosphorous containing polysulfates, oximes,
quinones, peroxides and others. The velocity of cross-linking may
be controlled by the use of cross-linking promoters and
accelerators. Since the first and second cross-linking agents may
be the same or very similar reagents, the uniform cross-linking
reaction and the gradient cross-linking reaction may be controlled
by selecting appropriate immersion conditions, such as
concentration of the ingredients, temperature and time of
immersion. The end products are disclosed as being useful as oil
seals, gaskets, O-rings, cable covers and other uses for nonsticky,
low friction articles. Since the elastomeric substrates disclosed
in this patent are non-water soluble, and thus, not swellable in
water, they are not useful in accordance with the present
invention.
There have been suggestions for uses of collagen in ophthalmology
but the focus has been on the problems of reabsorption and optical
clarity. Balazsl, "New and Controversial Aspects of Retinal
Detachment", A. McPherson, editor, page 371, Harper, New York
(1968), injected hyaluronic acid and a mixture of hyaluronic acid
and collagen (reconstituted vitreous) into the vitreous cavities of
owl monkeys. With reconstituted vitreous, less transparency was
noted, and upon injection into the eye the solution gelled and did
not mix with water. In studies on 50 owl monkeys, both the
hyaluronic acid and the vitreous disappeared in four to five months
and were replaced by the animals' own vitreous. Dunn, et al., Surg.
Forum 19, 492 (1968); and Rubin, et al., Tech. Rev. 71 (Number 2)
(1969) used gels made from enzyme-treated collagen as a vitreous
replacement. They found that gels could be stabilized by reducing
agents such as ascorbic acid. Ultraviolet irradiation was used to
crosslink the gel and increase stability. Rubin, et al. also used
extruded collagen gels as implants to relieve glaucoma. However, no
details of the preparation or procedures were given.
There have been some studies for the uses of collagen in a drug
delivery system. It is assumed that collagen-formed complexes with
various substances of varying stability dissociate slowly when
administered subcutaneously, intramuscularly and intraperitoneally,
therefore prolonging the pharmacological action of the drug. See
Horakova, et al., Therapie 22, 1455 (1967). Hardy, U.S. Pat. No.
3,469,003, injected patients with vaccines and other drugs using
reconstituted collagen as a vehicle. The effectiveness of local
anesthetics such as procaine and lidocaine, analgesics (pethidine)
and neuroplagique (perathiapine) was extended three to five times
when compared with controls injected with the drug alone. However,
this observation is possibly explained by retardation of the
diffusion of the drug into the systemic circulation by the viscous
collagen-drug complex.
However, it is believed that there has not been a suggestion in the
art to form collagen-shaped articles having a surface which is
crosslinked below such surface in a decreasing concentration
gradient of cross-linking sites in order to control the release
rate of a drug within the device.
Moreover, there has been no suggestion to surface crosslink
microspheres of collagen for use as controlled release drug
delivery vehicles.
It is thus an object of the present invention to provide a method
for forming collagen-shaped articles which are crosslinked, at
least on one surface thereof, to control the release of the drug
absorbed into the collagen from that surface.
It is another object of the present invention to provide ophthalmic
inserts of collagen which are crosslinked at one surface thereof to
retard dehydration and drug release through that surface, but
uncrosslinked at the opposite surface, to promote drug release.
It is yet another object of the present invention to provide
collagen microspheres, which are crosslinked to a depth having a
decreasing concentration gradient, which not only controls and
retards diffusion of the drug from the microspheres but also
controls and retards bioerosion of the microsphere.
These and other objects of the present invention will be apparent
from the following description, the appended claims and from the
practice of the invention.
SUMMARY OF THE INVENTION
The present invention provides a method for forming a controlled
release polymeric substrate comprising the steps of contacting a
water-swellable polymeric substrate or a portion thereof, with a
mixture containing a cross-linking agent in a liquid carrier
comprising water and an organic liquid for a period of time, at a
temperature and at a concentration of the agent sufficient for the
agent to penetrate the surface of the substrate and form
cross-linking bridges in the substrate in a decreasing
concentration gradient beneath the surface. The concentration of
the organic liquid in the liquid carrier is sufficient to prevent
dissolution of any significant amount of the substrate while in
contact with the liquid carrier.
The crosslinked substrate is then preferably loaded with the
bioactive agent by soaking in a solution of the bioactive agent.
The method is particularly adaptable for preparing microspheres of
crosslinked substrates which contain the bioactive agent. Upon
contact of the loaded microspheres with body fluids or tissues the
microspheres are gradually eroded by the action of enzymes or other
active agents in the body fluids or tissues, or impregnated within
the microspheres.
BRIEF DESCRIPTION OF THE DRAWINGS
In the accompanying figures:
FIG. 1 is a graph of the swell gradient for crosslinked collagen
gels as a function of reaction time of the gel with the
cross-linking agent EDC.
FIG. 2 is a graph of the swell gradient for crosslinked collagen
gels as a function of EDC concentration.
FIG. 3 is a graph of the swell gradient of crosslinked collagen
gels as a function of acetone concentration in the reaction
solvent.
FIG. 4 is a graph of the time release profile of pilocarpine from
microspheres made according to Example 6.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
According to the present invention there is provided a method for
forming a controlled release polymeric substrate which is
crosslinked on at least one surface thereof in a decreasing
concentration gradient of crosslinks beneath the surface. The
cross-linking is performed with a cross-linking agent which is
contacted with the substrate in the presence of a liquid carrier
comprising water and an organic liquid. The cross-linking agent
should be at least partially soluble in the solvent mixture of the
organic liquid and water.
The substrate which may be crosslinked according to the present
invention to form controlled release articles include
water-swellable proteinaceous material such as collagen, gelatin,
elastin, albumin, chitosan (deacetylated chitin), and other
polymeric materials which have functional moieties such as amino
groups, carboxyl groups and hydroxyl groups which can serve as
handles for the cross-linking reaction. Other substrates include
polymeric materials having hydroxyl and carboxyl functionalities as
functional handles for cross-linking such as glycocyamine
glycosoamino glycans (hyaluronic acid, chondroitin sulfate,
dermatin sulfate, heparin, etc.); polyacrylic acid; cellulose and
cellulose derivatives; alginate polymers; and amino functional
polymers, carboxyl or hydroxyl functional polymers such as hydroxyl
and carboxyl functional polymers listed above combined with amino
functional polymers such as chitosan or protein such as collagen.
The preferred substrates are collagen in any of its natural or
modified forms.
Bioactive agents include, but are not limited to antibiotics,
analgesics, anti-inflammation agents, anti-glaucoma agents,
vaccines, anti-neoplastic agents, etc. Representative anti-glaucoma
agents are Timolol, Epinephrine, Betaxolol, Pilocarpine, and
Carbonic Anhydrase Inhibitors. Representative antibiotics are
Tobramycin, Gentamycin, and Ciprofloxcin. Analgesics,
anti-neoplastic agents and vaccines include Cidocaine,
Proparacaine, 5-FU, Methotrexate, and inactivated viruses. These
bioactive agents may be used alone, or in combination, depending
upon the desired therapy. The amount of bioactive agent
incorporated into the substrate will depend upon the surface area
and volume of the substrate, the period of contact of the substrate
with the bioactive agent containing solution during loading of the
substrate, and concentration of the solution. Incorporation of the
desired dosage of bioactive agent into the substrate may be readily
achieved by variation of one or more of these parameters. The
substrate is typically loaded by soaking in a solution of the
bioactive agent, preferably after the substrate has been
cross-linked. In some instances, the substrate may be loaded before
cross-linking, recognizing that some of the bioactive agent may be
lost during the subsequent cross-linking process.
The cross-linking agent should be at least partially soluble in the
water and organic solvent mixture. The preferred reagents are the
water-soluble carbodiimides, in particular
1-ethyl-3-(3-diethylaminopropyl) carbodiimide (EDC) and
1-cyclohexyl-3-(2-(3'-morpholino)-ethyl) carbodiimide (CMC).
Preferably, these agents are used at an initial concentration in
the solvent in the range of about 0.1 to 10 mg/ml.
The solvent mixture in which the cross-linking reaction is
conducted is preferably a mixture of water and an organic
water-miscible solvent such as a ketone, alcohol, ester, etc. The
preferred organic solvent is acetone. Representative organic
solvents include alcohols such as methanol and ethanol, ketones
such as acetone and methylethyl ketone, esters such as ethyl
acetate, carbon disulfide and mixtures thereof.
The substrate is water-swellable so that when it is in contact with
the solvent containing the cross-linking agent, the water content
in the solvent will cause the substrate to swell thereby
facilitating penetration of the cross-linking agent into the
substrate. Generally the liquid mixture used to contact the
substrate for cross-linking will comprise from 5% to 95% by volume
of the organic solvent, with the remaining being water. The organic
solvent preferably limits the penetration of the cross-linking
agent, which is preferably water-soluble. Therefore, by increasing
the organic solvent content in the mixture, the depth of the
cross-linking agent penetration into the substrate can be reduced.
By control of the time of contact of the substrate with the
swelling solvent and the cross-linking agent, the temperature,
concentration of agent and concentration of organic solvent in the
mixture, the depth of penetration of the cross-linking agent into
the substrate can be controlled, as well as the crosslinked
density, which is continuously decreasing from the surface of the
substrate to the point at which there is no cross-linking. For
example, when the shape of the collagen device is a thin film, such
as for an ocular bandage, which itself is only about 100 microns
thick, it may be desirable to crosslink one surface (the outer
surface) of the bandage and to have a gradual gradient of
decreasing cross-linking density into the thickness of the film
achieving a cross-linking density of zero before penetration to the
opposite (inner) surface. In this manner the outer surface of the
ocular bandage will have the highest density of cross-linking
thereby minimizing the loss of bioactive agent and moisture through
the outward facing surface, while still allowing the release of the
bioactive active agent through the uncrosslinked inner surface
facing the retina where the bioactive agent is most needed. The
uncrosslinked surface also advantageously provides a hydrated,
cushioning surface against the eye.
In another embodiment the collagen may be formed into microspheres,
then impregnated with the bioactive agent and an enzyme which
degrades collagen, such as Collagenase or Cathepsin. The
microspheres may be formed and preserved in a relatively dry state
in which the enzyme is inactive. Upon exposing the microspheres to
body tissues or fluids, the collagen will hydrate and swell, and
the enzyme will be activated. However, since the area in which the
least amount of cross-linking is present is at the interior of the
microsphere, degradation will occur the fastest in the interior of
the microsphere. The last portion of the microsphere to degrade
will be the outer surface or shell where there is maximum
cross-linking. Upon penetration of the shell by degradation, the
microsphere will collapse and the remaining bioactive agent will be
released.
The cross-linking agent concentration is generally in the range of
0.01 to 5 percent by weight in the aqueous:organic solvent mixture.
Cross-linking accelerators may be utilized, as appropriate. The
temperature of immersion is preferably in the range of about
4.degree. C. to about 50.degree. C. and the time of immersion is
appropriately selected within the range of about 5 seconds to about
72 hours. A useful period of immersion is from 0.05 to 24
hours.
After the immersion, the article is removed from the treating
solution and preferably soaked in purified water. The substrate may
be treated with the bioactive agent before or after cross-linking,
as appropriate. For example, if the collagen is in the shape of a
microsphere the collagen will first be treated with a bioactive
agent and other agent, such as an enzyme, which is to be retained
within the microsphere, then the outer surface of the microsphere
is crosslinked in accordance with the present invention.
The invention is illustrated by the following examples which are
not intended to limit the invention in any way.
EXAMPLE 1
A collagen dispersion (2% bovine hide source) was prepared at pH
3.8, and added to 13.times.100 mm borosilicate test tubes in such a
manner as to produce 13.times.10 mm cylindrical collagen gels. To
each test tube was added 8ml of an appropriate cross-linking system
which contained EDC in an acetone/water solvent mixture. Before the
reaction was quenched, the cylindrical collagen gels were removed
from the tubes and sliced into two nearly equal segments
(approximately 13.times.5 mm). The top segment and the bottom
segment were then placed into two different test tubes. Each test
tube contained 8ml of acetic acid (quenching agent). After 18
hours, the swelled segments were weighted, wet and then dry. The
inverse of swell was evaluated as a function of the following
parameters: 1) reaction time, 2) EDC concentration, and 3) the
ratio of acetone to water in the reaction solvent. The inverse of
swell was evaluated because it is relevant to the actual crosslink
density. The inverse of the dry volume fraction of a polymer in a
solvent is proportional to the crosslink density of the polymer. By
varying the extent of reaction time while holding the concentration
of EDC at 20 mg/ml and the ratio of acetone to water in the
reaction solvent at 9/1, matrices with measurable crosslink
gradients were produced. The inverse of the swell of the matrices
was plotted as a function of the extent of reaction time (FIG. 1).
The inverse of the swell for the top slice, the bottom slice, and
the average of both were plotted. Each data point was then averaged
for 10 samples. There was a significant difference in swell from
the top of it to the bottom of each piece tested after 1.5, 3.0 and
6.0 hours. Crosslink gradient can be controlled by limiting the
extent of reaction through the gel. If the reaction is quenched
before the cross-linking system diffuses to the entire gel, the
part of the gel which has not come into contact with the
cross-linking system will have a lower crosslink density than the
parts in the gel which were in contact with the cross-linking
system. The longer the gel remains in contact with the
cross-linking system, the faster the matrix will approach a uniform
crosslink density.
EXAMPLE 2
Concentration of EDC was increased from 5 to 80 mg/ml while holding
the reaction time at 6 hours and the ratio of acetone to water in
the reaction solvent at 9/1, the swell gradient from top to bottom
decreased. See FIG. 2. The reaction proceeded more rapidly as the
concentration of EDC was increased. This shows that one may
increase the crosslink density gradient for materials crosslinked
at higher EDC concentrations by reducing the extent of reaction
time or limiting the diffusion of the cross-linking system.
EXAMPLE 3
To evaluate the effect of solvent systems, the swell was monitored
and plotted as a function of the ratio of acetone to water in the
reaction solvent while holding the concentration of EDC at 20 mg/ml
and the extent of reaction time at 6 hours. Examination of FIG. 3
indicates that a ratio of 1/3 acetone to water in the reaction
solvent creates a significant difference in the crosslink density
from the top to the bottom of the collagen gels tested. As the
acetone to water ratio was increased to 3/1, there was no longer
any observable difference in swell from top to bottom. When the
acetone to water ratio was increased to 9.5/0.5, the difference in
crosslink density from top to bottom reappeared. This may be
explained by the following. At higher water concentrations the
hydrolysis of the activated ester and EDC impedes the desired amide
bond formation needed to crosslink the matrix. Therefore as the
solvent front proceeds through the gel the amount of EDC available
for cross-linking is decreased (due to hydrolysis) thereby forming
a crosslink density gradient. As the acetone concentration was
increased to greater than 75 percent, the diffusion of the
cross-linking system was slowed, which in turn may have caused the
observed gradient at 95 percent acetone.
EXAMPLE 4
Preparation of Hydrogel Micro spheres
Two grams of collagen was dissolved into 100 ml of distilled water
with gentle agitation. After the collagen had completely dissolved
the pH of the solution was adjusted to 6.0 with a solution of
sodium hydroxide or hydrochloric acid. Two grams of poly-acrylic
acid (Carbopol 934 P, Goodyear) was dissolved into 100 ml of
distilled water with gentle agitation. After the poly-acrylic acid
had completely dissolved the pH of the solution was adjusted to 6.0
with a solution of sodium hydroxide or hydrochloric acid.
Five grams of the collagen solution was combined with 5 grams of
acrylate solution and diluted with 10 ml of distilled water. To
this solution, 4 grams of sorbitan monooleate was added and the
resulting mixture was thoroughly mixed. Using a high shear mixer,
25 ml of toluene was slowly added and an emulsion was formed. The
resulting emulsion was poured into 8 volumes of acetone with gentle
mixing. A dispersion of microspheres was formed. A solution of 15
mg of ethyldimethylaminopropyl carbodiimide (EDC) in 1 ml of water
and 9 ml of acetone was added to the dispersion. The mixture was
allowed to react for 1 hour.
The microspheres were isolated by centrifugation. The isolated
microspheres were resuspended into 30 ml of acetone and isolated by
centrifugation. This procedure was repeated 2 more times. The
acetone washed microspheres were suspended in 40 ml of 5 mM acetate
buffer (pH=5) and then isolated by centrifugation. The procedure
was repeated 2 time substituting distilled water for acetate
buffer. Two ml of water were added to the isolated microsphere to
yield a concentrated slurry of microspheres.
EXAMPLE 5
Drug Loading of Hydrogel Microspheres
Three hundred thirty ul of concentrated slurry, prepared as
described in Example 4, was added to 670 ul of a 7.5% pilocarpine
solution. The solution was gently mixed for 5 min. The resulting
solution contains drug loaded microspheres. The amount of the
pilocarpine incorporated into the microspheres was determined by
measuring the concentration of pilocarpine in the aqueous portion
of the microsphere dispersion and comparing that to the amount of
pilocarpine added to the solution, the difference being the amount
incorporated in to the microsphere.
EXAMPLE 6
Ocular Model In-Vitro Release of a Drug from Microspheres
A dispersion of drug loaded microspheres, prepared as described in
Example 5, was placed into a 500 ul reservoir with 2 openings on
opposite sides was filled with an aqueous solution of drug. The
reservoir contained a 0.45 um filter between the two openings. The
inlet was connected to a peristaltic pump via silicone tubing. The
reservoir was perfused with physiological saline solution and the
eluate was collected. The rate of perfusion was adjusted such that
the rate of elimination of drug from the reservoir was comparable
to the rate of elimination of a drug from the tear film of the eye.
The concentration of drug in the eluate was determined by UV
spectroscopy and graphed versus time, shown in FIG. 4.
EXAMPLE 7
Preparation of Hollow Spheres
A mixture of 230 ml of acetone, 20 ml of RO water, and 125 mg of
ethyl-(dimethylaminopropyl)carbodiimide hydrochloride is made in a
250 ml graduated cylinder. The solution was mixed thoroughly. A two
percent dispersion of hydrolyzed bovine tendon collagen (SEMEX II)
at pH 3.8 was then added to the graduated cylinder dropwise with a
7.5 ml polyethylene disposable transfer pipet. Approximately 30
drops of collagen dispersion were added to the reaction mixture.
After intervals of five, ten, and twenty minutes, ten of the
collagen drops were removed, and placed in a vial containing RO
water. After soaking in RO for twenty minutes, each vial containing
the crosslinked collagen material collected at each time interval
was photographed. A crosslinked droplet of collagen was then
removed from each vial, and dissected into three slices, so that
the center slice formed a doughnut shape. The cross-section of the
center slices for each time interval was magnified and
photographed.
The photographs showed that the spheres were hollow. In addition,
the time progression results show that the thickness of the shell
can be controlled by varying the crosslinking time. As the
crosslink density of a collagen matrix increases the percent
hydration of the collagen matrix decreases. Thus, the collagen
matrices become less dense than water as the crosslink density is
increased.
* * * * *